Journal: Scientific Reports

Article Title: Characteristics and time points to inhibit ferroptosis in human osteoarthritis

doi: 10.1038/s41598-023-49089-y

Figure Lengend Snippet: Ferroptosis occurs in OA cartilage in a region-specific manner. ( A ) Representative immunohistochemical (IHC) staining of NC (Negative Control), GPX4, ACSL4, MMP13, and COL2A1 in paired intact and damaged human OA cartilages. Scale bars: 100 μm (top) and 20 μm (bottom). ( B ) Quantification of immunohistochemical analysis (n = 3). ( C ) RT-qPCR was used to detect the mRNA expression of MMP13, COL2A1, GPX4, ACSL4 in human OA cartilage (n = 3). ( D ) Representative immunofluorescence staining and quantification analysis of GPX4 in human OA cartilage (n = 3). Scale bar: 50 μm. ( E ) Representative immunofluorescence analysis of ACSL4 in human OA cartilage (n = 3). Scale bar: 50 μm. ( F ) JC-1 staining was performed to monitor mitochondrial membrane potential (Δψm). The ratio of the red/green fluorescence indicated the degrees of mitochondrial damage (n = 3). Scale bar: 50 μm. Data are expressed as means + SD. For all experiments p-values calculated by unpaired two-tailed Student’s t test, p < 0.05, a statistically significant difference.

Article Snippet: After incubation with primary antibodies against COL2A1 (1:100; Servicebio, GB11021), MMP13 (1:100; Servicebio, GB11247), GPX4 (1:50; Zen-bio, R24461), and ACSL4 (1:20; Proteintech, 22401-1-AP), the sections were incubated with secondary antibody (Servicebio, G1215), followed by counterstaining with hematoxylin and visualized by DAB (Servicebio, G1212).

Techniques: Immunohistochemical staining, Immunohistochemistry, Negative Control, Quantitative RT-PCR, Expressing, Immunofluorescence, Staining, Membrane, Fluorescence, Two Tailed Test

Journal: Scientific Reports

Article Title: Characteristics and time points to inhibit ferroptosis in human osteoarthritis

doi: 10.1038/s41598-023-49089-y

Figure Lengend Snippet: Ferroptosis increased with increasing cartilage degeneration in human OA. ( A ) Safranin O-fast green (S.O) staining of OA cartilages. ( B ) Modified Mankin score of OA cartilage. ( C ) Representative IHC staining of NC (Negative Control), GPX4, ACSL4, MMP13, and COL2A1 in human OA cartilages. Scale bars: 100 μm (top) and 20 μm (bottom). ( D ) Quantification of IHC analysis (n = 3). ( E ) RT-qPCR was used to detect the mRNA expression of MMP13, COL2A1, GPX4, ACSL4 in human OA cartilage. (n = 3). ( F ) MDA contents of human OA cartilage (n = 3). ( G ) Flow cytometry analysis of lipid peroxide level of human OA cartilage. ( H ) Representative immunofluorescence staining and quantification analysis of GPX4 in human OA cartilage (n = 3). Scale bar: 50 μm. ( I ) Representative immunofluorescence staining and quantification analysis of ACSL4 in human OA cartilage (n = 3). Scale bar: 50 μm. ( J ) Mitochondrial membrane potential was detected by JC-1 assay (n = 3). Scale bar: 50 μm. Data are expressed as means + SD. For all experiments p-values calculated by ANOVA analysis, p < 0.05, a statistically significant difference.

Article Snippet: After incubation with primary antibodies against COL2A1 (1:100; Servicebio, GB11021), MMP13 (1:100; Servicebio, GB11247), GPX4 (1:50; Zen-bio, R24461), and ACSL4 (1:20; Proteintech, 22401-1-AP), the sections were incubated with secondary antibody (Servicebio, G1215), followed by counterstaining with hematoxylin and visualized by DAB (Servicebio, G1212).

Techniques: Staining, Modification, Immunohistochemistry, Negative Control, Quantitative RT-PCR, Expressing, Flow Cytometry, Immunofluorescence, Membrane

Journal: Scientific Reports

Article Title: Characteristics and time points to inhibit ferroptosis in human osteoarthritis

doi: 10.1038/s41598-023-49089-y

Figure Lengend Snippet: Inhibition of ferroptosis can improve chondrocyte function in mild OA, but not moderate and severe OA. ( A ) The effects of Fer-1 (2 μM) on the viability of human normal chondrocytes after 24 h of treatment was determined by CCK-8 assay. ( B ) Human chondrocyte viability was determined by CCK-8 assay. ( C ) The viability of chondrocytes was determined by CCK-8 assay following treatment of chondrocytes of each group with Fer-1 (2 μM) for 24 h. ( D – I ) The mRNA expression levels of MMP13, COL2A1, GPX4, ACSL4, SLC7A11 and P53 were detected by RT-qPCR following treatment of chondrocytes of each group with Fer-1 (2 μM) for 24 h. ( J ) Representative immunofluorescence staining and quantification analysis of GPX4 in the chondrocytes of each group treated with Fer-1 (2 μM) for 24 h. ( K ) Representative immunofluorescence staining and quantification analysis of ACSL4 in the chondrocytes of each group treated with Fer-1 (2 μM) for 24 h. ( L ) Mitochondrial membrane potential was detected by JC-1 assay following treatment of chondrocytes of each group with Fer-1 (2 μM) for 24 h. Scale bar: 50 μm. Data are expressed as means + SD. For all experiments p-values calculated by unpaired two-tailed Student’s t test, p < 0.05, a statistically significant difference.

Article Snippet: After incubation with primary antibodies against COL2A1 (1:100; Servicebio, GB11021), MMP13 (1:100; Servicebio, GB11247), GPX4 (1:50; Zen-bio, R24461), and ACSL4 (1:20; Proteintech, 22401-1-AP), the sections were incubated with secondary antibody (Servicebio, G1215), followed by counterstaining with hematoxylin and visualized by DAB (Servicebio, G1212).

Techniques: Inhibition, CCK-8 Assay, Expressing, Quantitative RT-PCR, Immunofluorescence, Staining, Membrane, Two Tailed Test